Photosynthetic production of 3-hydroxybutyrate from carbon dioxide

ABSTRACT

Construction and expression of synthetic pathways to produce (S) or (R)-3-hydroxybutyrate (3HB) as enantiomerically-pure products by genetically engineering cyanobacterium  Synechocystis  sp. PCC 6803. Under optimized growth conditions, the pathway employing phaA and phaB from  R. eutropha  was the most effective, producing up to 533.4±5.5 mg/l (R)-3HB after 21 days photosynthetic cultivation. For the first time, the feasibility and high efficiency of producing 3HB using solar energy and CO 2  as sole energy and carbon sources by engineered cyanobacteria is demonstrated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.14/479,893, filed on Sep. 8, 2014, which claims the benefit of U.S.Provisional Patent Application No. 62/017,650, filed on Jun. 26, 2014,and is a continuation of International Application No.PCT/US2013/029997, filed on Mar. 8, 2013, which claims the benefit ofU.S. Provisional Patent Application No. 61/646,807, filed on May 14,2012, the disclosures of each patent or patent application areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Although human society has progressed significantly over past centuriesthrough the development and use of petroleum-derived products (e.g.fuels, plastics, solvents, etc.), their over-utilization has causedenvironmental issues including increasing atmospheric concentration ofCO₂ (a greenhouse gas), pollution from petrochemical production and use,and disposal of non-biodegradable plastic materials. More importantly,petroleum resources are finite and not renewable in nature. For thesereasons it is necessary to seek alternative approaches to produce fuelsand chemicals using renewable resources. Photosynthetic cyanobacteriahave attracted significant attention in recent years as a ‘microbialfactory’ to produce biofuels and chemicals due to their capability toutilize solar energy and CO₂ as the sole energy and carbon sources,respectively.

Lipid-rich cyanobacteria and microalgae have most notably been employedto produce fuels such as biodiesel. Cyanobacteria are also naturalproducers of the naturally-occurring biodegradable plasticpoly-β-hydroxybutyrate (PHB). Despite efforts to enhance PHBbiosynthesis through both genetic engineering strategies and theoptimization of culture conditions, PHB biosynthesis by cyanobacteriawas a multi-stage cultivation process that involved nitrogen starvationfollowed by supplementation of fructose or acetate, which does notcapitalize on the important photosynthetic potential of cyanobacteria.Most importantly, as neither lipids nor PHB are secreted by the cells,the required processes for their extraction are energy-intensive andremain as one of the major hurdles for commercial applications. As aresult, researchers have recently focused on engineering cyanobacteriato instead produce secretable biofuels and chemicals. However, mostproduction titers are below 200 mg/l and to our knowledge no reportdemonstrated the potential of employing photosynthetic microorganisms ina continuous production process.

SUMMARY OF THE INVENTION

Different from PHB, which accumulates inside cells, 3-hydroxybutyrate(3HB) is a small molecule that could possibly be secreted out of thecells into extracellular environment, thereby facilitating itscollection. 3HB can then be chemo-catalytically polymerized to producePHB or be co-polymerized with other organic acid compounds to synthesizerenewable plastics with a broader range of chemical and materialproperties (including adjustable molecular weight and improved purity)relative to naturally-synthesized PHB. (R)- or (S)-3HB can also serve asa precursor for many stereo-specific fine chemicals such as antibiotics,pheromones and amino acids. Moreover, (R)-3HB has been found to be anadvanced nutrition source for tissue cells and can reduce the death rateof the human neuronal cells, improve mice memory and promote growth ofosteoblasts.

3HB synthetic pathways in cyanobacterium Synechocystis sp. PCC 6803(hereafter Synechocystis 6803) were constructed and demonstrated highlyefficient photosynthetic production and secretion of 3HB using solarenergy and CO₂ as the sole carbon and energy sources. Thus, multi-cycleor continuous production of 3HB from engineered Synechocystis arepossible.

Various other purposes and advantages of the invention will become clearfrom its description in the specification that follows. Therefore, tothe accomplishment of the objectives described above, this inventionincludes the features hereinafter fully described in the detaileddescription of the preferred embodiments, and particularly pointed outin the claims. However, such description discloses only some of thevarious ways in which the invention may be practiced.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of (S)-3HB and (R)-3HB biosynthesisfrom CO₂ in engineered Synechocystis. Thil, thiolase from C.acetobutilicum ATCC824; PhaA, thiolase from R. eutropha H16; PhaA2(Slr1993), native thiolase in Synechocystis 6803; Hbd(S)-3-hydroxybutyryl-CoA dehydrogenase from C. acetobutilicum ATCC824;PhaB, (R)-3-hydroxybutyryl-CoA dehydrogenase from R. eutropha H16; PhaB2(Slr1994), native (R)-3-hydroxybutyryl-CoA dehydrogenase inSynechocystis 6803; TesB, thioesterase II from E. coli XL1-Blue MRF′;PhaEC, native PHB polymerase in Synechocystis 6803.

FIG. 2 is a schematic representation of the modification ofSynechocystis chromosome for 3HB production. Site 1, the site on thegenome of Synechocystis 6803 between slr1495 and sll1397; Site 2, thesite between slr1362 and sll1274; Site 3, the site between slr1828 andsll1736 for phaEC deletion; Site 4, the site between slr1992 and phaA2(slr1993).

FIG. 3 depicts the cell density of different Synechocystis strainscultivated in shaking flasks.

FIG. 4 depicts extracellular production of 3HB by Synechocystis 6803 andengineered derivatives. 3HB titers were symbolized as grey bars andacetate titers were symbolized as yellow bars.

FIGS. 5A-C depict 3HB productivity for unit density of SynechocystisTAB1 cell cultures subjected to nitrogen starvation and normal BG11medium (control) cultures. 3HB production by the normal BG11 mediumcontrol dramatically increased by day 3. Culture grown under low lightintensity (LL, ˜45 μE/m²/s), middle light intensity (ML, ˜60 μE/m²/s)and high light intensity (HL, ˜90 μE/m²/s) were studied.

FIGS. 6A-C depict Synechocystis TAB1 cells subjected to nitrogenstarvation and normal BG11 medium (control) cultures. Overall, higher3HB production and cell density of the control versus that of thenitrogen-starved counterparts was observed.

FIG. 7 depicts the effect on 3HB production by nutrient supplementation.When the indicated amount of nutrient was supplemented into the cultureafter day 7, both biomass and 3HB production started to increase again.

FIGS. 8A-B depict biomass and 3HB production curves of Synechocystisstrain TABd under different nutrient supplementation conditions. (A)Biomass curves. Grey squares indicate non-N-supplementation; Opensquares indicate 5% N-supplementation; solid squares indicate 10%N-supplementation. (B) 3HB production curves. Grey triangles indicatenon-N-supplementation; Open triangles indicate 5% N-supplementation;solid triangles indicate 10% N-supplementation.

FIG. 9 depicts, after 10 days culture, chlorosis in the non-N and 5%-N(shown) cultures but not in the 10%-N cultures.

FIGS. 10A-B depict biomass and 3HB production curves of Synechocystisstrain TABd under 5% N-supplementation condition for 3HB production bymedium exchange.

FIG. 11 depicts continuous production of 3HB directly from atmosphericCO₂ by Synechocystis strain TABd. Solid triangles indicate 3HB titers;solid squares indicate cell densities represented by OD₇₃₀.

FIG. 12 is a schematic representation of (R)-3HB biosynthesis from CO₂in engineered Synechocystis.

FIG. 13 is a schematic structure of the five promoters.

FIGS. 14A-D are characterizations of the five promoters. (A) Celldensity of strains expressing tesB using each promoter. (B) Productionof (R)-3HB by each strain. (C) tesB mRNA abundance in each engineeredstrain. (D) Acetate accumulation in the culture medium of eachengineered strain.

FIGS. 15A-E are characterizations of the dual promoter system in strainPTrK16. (A) Schematic representation of the dual promoter system. (B)Cell densities for strains TTrK and SPTrK16 after growing under a lightintensity of 60 μE/m²/s for 5 days. (C) Production of (R)-3HB andacetate by strains TTrK and PTrK16. (D) tesB mRNA relative abundance onday 3.5. (E) Thioesterase activity analysis for TesB.

FIGS. 16A-B are (R)-3HB productions under different illuminationconditions. (A) (R)-3HB and acetate produced by strains TTrK and TABd(indicated with stars). (B) (R)-3HB and acetate produced by strainPTrK12.

FIGS. 17A-B are a performance of Synechocystis strains with two copiesof tesB and phaB. (A) Cell growth curve of strains TTrK, TTB2K3 andABdTB. (B) Production of (R)-3HB and acetate.

FIG. 18A-D are optimizations of the RBS for gene phaB1 in Synechocystisstrain R154. (A) The original and optimized ribosome binding site. (B)Acetoacetyl-CoA reductase activity. (C) Cell growth curve of strainsTTrK and R154. (B) Production of (R)-3HB and acetate.

FIGS. 19A-B show photosynthetic production of (R)-3HB from CO₂ bySynechocystis strain R168. (A) Cell growth curve of strains R168 andABdTB. (B) Time course of in-flask (R)-3HB generated by strains R168 andABdTB.

FIG. 20 is an in-flask and cumulative titers of (R)-3HB generated bystrain R168.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, this disclosure relates to engineered strains ofSynechocystis. 3HB synthetic pathways in cyanobacterium Synechocystis6803 were constructed and demonstrated highly efficient photosyntheticproduction of 3HB using solar energy as the sole energy source.

In a second aspect, this disclosure relates to highly efficientphotosynthetic production of 3HB using bicarbonate or CO₂ as the solecarbon source by engineered Synechocystis.

In a third aspect, this disclosure relates to biosynthesis of 3HB in aprocess coupled with oxygenic photosynthesis in engineeredSynechocystis.

In a fourth aspect, this disclosure relates to highly efficientsecretion of hydrophilic 3HB molecules by engineered Synechocystiswithout overexpression of specific transporters.

In a fifth aspect, this disclosure relates to multi-cycle or continuousphotosynthetic production of 3HB from engineered Synechocystis.

In a sixth aspect, this disclosure relates to photosynthetic productionof 3HB from engineered cyanobacteria.

While the embodiments described below utilize Synechocystis, those ofordinary skill will appreciate that other cyanobacterial species may beengineered to produce 3HB following the strategies and geneticengineering guidance provided herein. Therefore, this disclosure is notlimited to 3HB production from Synechocystis but rather extends to 3HBproduction from all cyanobacteria capable of genetic manipulation of 3HBbiosynthesis pathways.

Construction and expression of synthetic pathways to produce (S) or(R)-3-hydroxybutyrate (3HB) as enantiomerically-pure products usingcyanobacterium Synechocystis sp. PCC 6803 was undertaken as describedbelow. However, this disclosure is not limited to the exact methods andmaterials described.

Synechocystis Strains and Culture Conditions.

A series of Synechocystis strains were constructed using markermodification and markerless modification methods. Synechocystis 6803 andits derivatives were grown in BG11 medium under a light intensity of 35μE/m²/s unless otherwise specified. For BG11 plates for Synechocystisgrowth, 10 mM TES (pH 8.2), 3 g/l thiosulfate and 1.5% agar wassupplemented before autoclaving. E. coli XL1-Blue MRF′ (Stratagene, LaJolla, Calif.) was used as host to construct and store all recombinantplasmids. All strains of E. coli were cultivated in Luria-Bertani (LB)medium at 37° C. Antibiotics were supplemented as appropriate at thefollowing concentrations: 100 ng/l ampicillin, 30 ng/μl kanamycin, and25 ng/μl chloramphenicol. Bacillus subtilis strain 168 was obtained fromAmerican Type Culture Collection (ATCC) and was cultured in LB medium at30° C.

Synechocystis 6803 genomic DNA was purified by DNeasy Blood & Tissue Kit(QIAGEN, Valencia, Calif.) and subsequently used as template for PCRamplification of SR12 (slr1495) and SL12 (sll1397) DNA fragments. SR12and SL12 were recombined together by overlapping PCR and were insertedinto the SacI and KpnI restriction sites of the plasmid pBluescript IISK(+) (Stratagene, La Jolla, Calif.) to construct pBS-SRSL. From thegenomic DNA of Clostridium acetobutylicum ATCC 824, thil gene was PCRamplified using primers Th5 and Th8. The purified product was then againPCR amplified by primers Ptac and Th8 to construct Ptac-thil, whereinthil was under the control of the Ptac promoter. The gel-purifiedPtac-thil product was then again PCR amplified using primers TAC5 andTh8, the product of which was purified and restriction digested beforebeing inserted into the BamHI and SalI sites of pBS-SRSL to constructpBS-SPT. Next, hbd of C. acetobutylicum was PCR amplified with primersHBD3 and HBD6. The resultant fragment was purified and restrictiondigested before being inserted between the NcoI and SalI sites ofpBS-SPT to construct pBS-SPTH. Two fragments of the cat (Cm^(R)) gene onpACYC 184 (New England Biolabs, Ipswich, Mass.) were amplified usingprimer pairs Cat3 and Cat4, Cat5 and Cat6, and then were recombined byoverlapping PCR using primers Cat3 and Cat6 to remove the NcoIrestriction site in the open reading frame. The NcoI-removed cat genewas then inserted between the PstI and BamHI sites of pBS-SRSL toconstruct pBS-SCat. The Ptac-thil-hbd fragment of pBS-SPTH was PCRamplified using primers TAC5 and HBD6 and then inserted between theBamHI and SalI sites of pBS-SCat to construct pBS-SCPTH. The Ptac-thilfragment from pBS-SCPTH was PCR amplified using primers TAC5 and primerTh10 and was used to replace the original Ptac-thil fragment ofpBS-SCPTH between BamHI and NcoI to construct pBS-SCPTH2. The R.eutropha H16 gene phaB was PCR amplified with primers PHAB11 and PHAB12using pETphaAphaB (reconstructed based on the methods of Tseng et al. inconstructing pET-P-P) as template and was inserted between the MluI andHindIII sites of pBS-SCPTH2 to construct pBS-SCPTB. The gene phaA fromR. eutropha H16 was PCR amplified using primers PHAA11 and PHAA12 withpETphaAphaB as template. The purified product was then amplified usingprimers Ptac and primer PHAA12 to construct the Ptac-phaA fragment.Ptac-phaA was further PCR amplified using primers TAC5 and PHAA12 beforebeing inserted between the BamHI and MluI sites of pBS-SCPTB toconstruct pBS-SCPAB.

The DNA fragment containing GTP from Synechocystis 6803 was PCRamplified using primers GTP1 and GTP2 and was inserted between the SacIand PstI sites of pBS-SCat to construct pBS-SCG. The DNA fragment PHAUfrom Synechocystis 6803 was PCR amplified using primers PHAU1 and PHAU2before being further PCR amplified using primers Ptac and PHAU2 toconstruct Ptac-PHAU. Ptac-PHAU was then amplified using primers TAC5 andPHAU2 and the product was inserted between the BamHI and KpnI sites ofpBS-SCG to construct pBS-GCPU.

The DNA fragments SR56 and SL56 were PCR amplified using primer pairsSR5 and SR6 and SL5 and SL6 with Synechocystis 6803 genomic DNA astemplate. Fragments SR56 and SL56 were recombined together byoverlapping PCR before being inserted into the SacI and XhoI restrictionsites of the plasmid pBluescript II SK(+) to construct pBS-S2. pBS-S2was digested with MluI and SalI before being ligated with kan (Kan^(R))which was amplified from pET-30a(+) (Novagen, Madison, Wis.) usingprimers Kan1 and Kan2 to construct pBS-S2K. The E. coli gene tesB wasamplified with primers TESB1 and primer TESB2 using the E. coli XL1-BlueMRF′ genomic DNA as template. Ptac promoter was PCR amplified withprimers TAC11 and TACTESB1 using pBS-SPTH as template. The Ptac and tesBcontaining PCR products were then recombined by overlapping PCR usingprimers TAC11 and TESB2 to construct the fragment Ptac-tesB. Ptac-tesBwas digested with BglII and HindIII before being inserted between thecorresponding sites of pBS-S2K to construct pBS-SPtTeK.

The DNA fragment PpasD56 was PCR amplified from the Synechocystis 6803genomic DNA using primers PpsaD5 and PpsaD6. The thil gene was PCRamplified from C. acetobutylicum ATCC 824 genomic DNA using primers Th1and Th2. The PCR product was recombined with PpsaD56 by overlapping PCRusing primers PpsaD5 and Th2 and the resultant PpsaD-thil product wasinserted between the BamHI and MluI sites of pBS-S2K to constructpBS-SPTK. Ptac was amplified from pBS-SPTH using primers TAC5 andTAC-PTB3 and then inserted between the BamHI and NdeI sites of pBS-SPTKto construct pBS-SPtK. The sacB gene was PCR amplified using primersSACB8 and SACB9 using B. subtillus genomic DNA as template. The productwas restriction digested and inserted between the NdeI and MluI sites ofthe pBS-SPtK plasmid to construct pBS-SPSK2. DNA fragments PHA1 and PHA2were each PCR amplified from Synechocystis 6803 genomic DNA using primerpairs PHA11 and PHA12 and PHA21 and PHA22. Fragments PHA1 and PHA2 werethen recombined together by overlapping PCR using primers PHA11 andPHA22 to construct the DNA fragment PHA. PHA was then inserted betweenthe XhoI and SacI sites of pBS-S2 to construct pBS-PHA. ThePtac-sacB-kan fragment was removed from pBS-SPSK2 by digestion withBamHI and SalI and then inserted between the corresponding sites ofpBS-PHA to construct pBS-SPSK3.

Modification of Synechocystis Genome.

Synechocystis strains were grown to an OD₇₃₀ of 0.2-0.4, at which timepoint 0.5 ml culture was pelleted by centrifugation at 2700×g for 10 minat room temperature. The cell pellet was re-suspended in 50 μl freshBG11 medium to which approximately 2 μg of the chromosome-targetingplasmid was added and mixed. The mixture was incubated at 30° C. underlight (˜25 μE/m²/s) for 5 h before being plated on BG11 solid agarplates with appropriate antibiotics supplements, 10 ng/μl kanamycin or 5ng/μl chloramphenicol. The plates were placed at 30° C. under light andcolonies could be seen within two weeks. Individual colonies were thenisolated and re-streaked on BG11 solid agar plates with appropriateantibiotics for additional one to two weeks to achieve full chromosomesegregation, as was verified by colony PCR. Alternatively, markerlessmodification of the Synechocystis genome was conducted using the methoddescribed previously with minor modifications.

Briefly, fragment Ptac-sacB-kan was inserted into the neutral site ofSynechocystis 6803 using a marker modification method as described inthe text. After confirming that the resultant strain was genotypicallypure as verified using colony PCR, the strain were grown in BG11 mediumto an OD₇₃₀ of 0.2-0.4, when cells were centrifuged at 2700×g for 10 minat room temperature and was resuspended to OD₇₃₀ of 4.0 by 50 μl BG11.About 2 μg of chromosome-targeting plasmid pBS-PHA was added and mixedwell with the cells. The mixture was incubated at 30° C. under light (25μE/m²/s) for 5 h before being transferred into 25 ml BG11 medium in a 50ml flask. Cells were then further cultivated for 4-5 days after whichabout 1.3×10⁸ cells (assuming OD₇₃₀ of 0.6 equals to 10⁸ cells/ml) werespread onto a BG11 plate containing 4.5% (w/v) sucrose forcounter-selection. The plates were incubated at 30° C. under light forone or two weeks before colonies appeared. Individual colonies were thenre-streaked on fresh BG11 plates with 4.5% sucrose for additional one totwo weeks until full chromosome segregation was achieved, as verified bycolony PCR.

Gene Expression Analysis:

Synechocystis strains were inoculated in 50 ml flasks, each containing10 ml BG11 (10 mM TES-NaOH), to an initial OD₇₃₀ of 1.5. Then cells wereincubated in a shaking bed (150 rpm) at 30° C. with light intensity of35 μE/m²/s for 5 days. Every 24 h, 0.5 ml 1.0M NaHCO₃ was added to eachculture and the pH of the culture medium was adjusted to 7.5 by additionof 10 N HCl.

RT-qPCR.

Approximately 1.67×10 Synechocystis cells (assuming OD₇₃₀ of 0.6 equalsto 10⁸ cells/ml) were collected by centrifugation at 17,000×g, 4° C. for1 min. The supernatant was discarded and the cell pellet was kept under−80° C. until RNA extraction. Total RNA extraction, cDNA synthesis andRT-qPCR were conducted using methods described previously.

Enzyme Activity Assay.

3.3×10′ cells were collected by centrifugation at 5000×g at 4° C. for 10min. The supernatant was discarded and the cell pellet was used eitherimmediately or frozen at −80° C. for assaying at a later date. For allenzyme assays, the cell pellet was first re-suspended in 1.0 ml 100-mMTris-HCl (pH7.5) and then subjected to sonication in ice bath using aBranson Digital Sonifier Model 102C CE (Branson Ultrasonics, Danbury,Conn.) and Sonic Dismembrator Model 500 (Fisher Scientific, Waltham,Mass.) to lyse cells. The sonication program consisted of:3-sec-on/3-sec-off for 100 cycles. Cellular debris was removed bycentrifugation at 17,000×g at 4° C. for 10 min. The resultantsupernatant was used for enzyme assays.

The thiolase (encoded by phaA2, phaA or thil) activity was determinedusing acetoacetyl-CoA and CoA as substrates. The decrease in absorbanceat 303 nm was monitored as function of time and specific enzyme activitywas calculated by using a molar extinction coefficient of 14,000M⁻¹cm⁻¹. The activity of (R)-3-hydroxybutyryl-CoA dehydrogenase (encodedby phaB2 or phaB) was determined using acetoacetyl-CoA and NADPH assubstrates. The activity of (S)-3-hydroxybutyryl-CoA dehydrogenase(encoded by hbd) was determined using acetoacetyl-CoA and NADH assubstrates. The decrease in absorbance at 340 nm was monitored over timeand specific enzyme activity was calculated by using a molar extinctioncoefficient of 6,220 M⁻¹cm⁻¹. The thioesterase activity was determinedusing butyryl-CoA, decanoyl-CoA or acetyl-CoA as substrate and therelease of CoA was monitored at 412 nm by using5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, St. Louis,Mo.). The molar extinction coefficient was taken as 13,600.

Thioesterase (TesB) Activity Specificity Assay.

The thioesterase activities were examined using different acyl-CoAsubstrates including decanoyl-CoA (10 carbon acyl group), butyryl-CoA (4carbon acyl group) and acetyl-CoA (2 carbon acyl group). The release ofCoA was monitored at 412 nm by using 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB; Sigma-Aldrich, St. Louis, Mo.). The molar extinction coefficientwas taken as 13,600.

For TesB assay in Synechocystis, 20 OD₇₃₀·mL Synechocystis strain TESBcells was collected after 5 days cultivation. The cell pellet was firstre-suspended in 1.0 ml 100-mM Tris-HCl (pH7.5) and then subjected tosonication in ice bath using a Branson Digital Sonifier Model 102C CE(Branson Ultrasonics, Danbury, Conn.) and Sonic Dismembrator Model 500(Fisher Scientific, Waltham, Mass.) to lyse cells. The sonicationprogram consisted of: 3-sec-on/3-sec-off for 100 cycles. Cellular debriswas removed by centrifugation at 17,000×g at 4° C. for 10 min. Theresultant supernatant was used for enzyme assays using same molarconcentration of different acyl-CoA substrates.

For TesB assay in E. coli, strain XL1-Blue/pBS-SPtTeK cells wascollected after ˜10 hours cultivation in a 50 ml tube containing 10 mlLB medium at 37° C., 200 rpm. After 10 h cultivation, the OD₆₀₀ of E.coli XL1-Blue/pBS-SPtTeK and E. coli XL1-Blue/pBS-S2K was 5.3 and 4.6,respectively, and thus 3.8 ml and 4.4 ml culture was pelletted for eachbefore sonication. The cells were lysed using same method as describedabove. The resultant supernatant was used for enzyme assays using samemolar concentration of butyryl-CoA and acetyl-CoA as substrates. E. coliXL1-Blue/pBS-S2K was used as a control in these enzyme assays.

3HB Production.

Each strain was inoculated into 20 ml BG11 medium in a 50 ml flask at aninitial OD₇₃₀ of 0.1, and then grown photosynthetically to an OD₇₃₀ of0.5-1.0 before NaHCO₃ was then added to a final concentration of 50 mM.When the cell density reached an OD₇₃₀ of 1.0-2.0, cells were collectedby centrifuging at 5000×g for 10 min at 20° C. Cell pellets werere-suspended in 10 ml of fresh BG11 containing 50 mM NaHCO₃ in 50 mlflasks to a cell density of an OD₇₃₀ of 1.5. The pH of the medium wasadjusted to 7.5. The re-suspended cells were then incubated in a shakingbed (150 rpm) at 30° C. with light intensity of 35 μE/m²/s for 5 days.Every 24 h, 0.5 ml 1.0M NaHCO₃ was added to each culture and the pH ofthe culture medium was adjusted to 7.5 by addition of 10 N HCl. Allculture experiments were conducted in triplicate for each strain.

Nitrogen Limitation Test.

Synechocystis TABd was grown to an OD₇₃₀ of 1.0-2.0 as described abovebefore the cells were pelleted and collected. Cell pellets werere-suspended in 10 ml BG11 containing 10 mM TES-NaOH (pH8.0) and 50 mMNaHCO₃ in 50 ml-flasks with an initial cell density of OD₇₃₀ of 2.0. Theinitial pH of culture medium was adjusted to 7.5 by adding 10 N HCl.Once daily, 1 ml of the culture was sampled for analysis and replacedwith 1 ml fresh BG11 containing 500 mM NaHCO₃ and 1.5 g/l, 0.75 g/1l andnone of NaNO₃, as appropriate. Thus, 50 mM fresh NaHCO₃ and either 10%,5% or 0% of fresh NaNO₃ were added to the culture medium each day(corresponding to 10%-N, 5%-N or non-N culture, respectively).

Production of 3HB by Intermittent Medium Exchange.

After 10 days cultivation (Cycle I) using the 5%-N supplementationstrategy as stated above, Synechocystis TABd cells were collected andre-suspended in 10 ml fresh BG11-10N (10 mM TES-NaOH, pH8.0) medium thatcontained only 10% of the NaNO₃ content in typical BG11 medium tore-initiate cultivation in 50 ml flasks for a second 10 days (days 11through 20; called Cycle II). During this period, 250 μl of cell culturewas sampled for analysis each day. After sampling, 250 μl fresh BG11medium, 10 μl 37.5 g/1l NaNO₃ (equivalent to the nitrate content of 250μl BG11) and 250 μl 2.0 M NaHCO₃ were added back into the culture. Notethat since evaporative water losses from the culture were calculated tobe about 260 μl per day, the above supplementation protocol was used tomaintain the total culture volume. This protocol resulted in the dailyaddition of 5% of the NaNO₃ to the culture medium.

Photosynthetic 3HB production from CO₂ . Synechocystis was inoculatedinto a 125 ml flask containing 75 ml autoclaved BG11 (10 mM TES-NaOH)medium to an initial OD₇₃₀ of 0.2. The culture was placed at 30° C. withcontinuous illumination of 120 μE/m²/s and was bubbled with ambient air.The aeration rate was initially set as 75 ml/min. When the culture OD₇₃₀surpassed about 0.6, the aeration rate was then increased to 250 ml/min.Daily, 1 ml of culture was sampled and 1 ml 5-fold concentratedsterilized BG11 medium was added back into the culture until day 18.After day 18, 1 ml of culture was sampled but no BG11 medium was addedback into the culture. The experiments were conducted in duplicates.

Product Quantification.

Standard solutions of 3HB were prepared in water using(±)-3-Hydroxybutyric acid sodium salt. Samples of the culture mediumwere centrifuged at 17,000 xg for 2 min at room temperature and thesupernatant was collected for analysis of products on an 1100 seriesHPLC equipped with a refractive index detector (Agilent, Santa Clara,Calif.). Separation of metabolites was achieved using an Aminex HPX-87Hanion-exchange column (Bio Rad Laboratories, Hercules, Calif.). Themobile phase consisted of 5 mM H₂SO₄ at an initial flow rate of 0.55ml/min before immediately and linearly increasing to a final flow rateof 0.8 ml/min over 12 min, followed by an 8 min hold. The columntemperature was maintained at 35° C. throughout.

3HB is the precursor for synthesizing the biodegradable plasticspoly-3-hydroxybutyrate (PHB), as well as many chiral fine chemicals. Forthe first two steps in the constructed pathways, namely thiolase and3-hydroxybutyryl-CoA dehydrogenase, gene pairs from three differentbacterial sources were comparatively examined: native Synechocystisslr1993 (phaA2) and slr1994 (phaB2) for (R)-3HB, phaA and phaB fromRalstonia eutropha H16 for (R)-3HB, and thil and hbd from Clostridiumacetobutylicum ATCC824 for (S)-3HB. The final step of all pathwaysconsisted of thioesterase II (encoded by tesB) from Escherichia coli. Tofacilitate carbon flux towards 3HB, slr1829 and slr1830 (encoding PHBpolymerase) were deleted from Synechocystis to eliminate PHB production.

Construction of 3HB-Producing Strains.

A series of strains were constructed to systematically explore thephotosynthetic production of (S)- and (R)-3HB by engineeredSynechocystis (Table 1) using standard molecular biology protocols. Thegeneral strategy is presented in FIG. 1. The E. coli thioesterase IIencoded by tesB gene has been utilized in each scheme to directlyhydrolyze (S)- or (R)-3-hydroxybutyryl-CoA to generate (S)- or (R)-3HB,respectively. We first introduced the tesB gene from E. coli into thegenome of Synechocystis 6803 to construct strain TESB which was expectedto realize the conversion of (R)- or (S)-3-hydroxybutyryl-CoA tocorresponding 3-HB. To complete the 3HB biosynthesis pathways, we nextintroduced three different sets of operons into Synechocystis TESB toexpress both thiolase and 3-hydroxybutyryl-CoA dehydrogenase. Theresultant strains included HB5 and TAB1, which respectively harboredthil and hbd from C. acetobutylicum ATCC 824 or phaA and phaB fromRalstonia eutropha H16. A third strain TPU3 was constructed by placing aPtac promoter just upstream of the native slr1993 (phaA2)-slr1994(phaB2) operon to enhance its expression (FIG. 1).

The Ptac promoter has been reported as a strong promoter inSynechococcus and Synechocystis and was used to initiate high-levelexpression of isobutanol biosynthetic genes in Synechococcus. Here, thePtac promoter was used to express all of the 3HB pathway genes (FIG. 2).All foreign genes were integrated into the neutral sites of theSynechocystis genome where no effect was expected (FIG. 2).Additionally, because native PHB synthesis would compete with 3HBbiosynthesis for the intermediate (R)-3-hydroxybutyryl-CoA (FIG. 1), thenative operon harboring phaE (slr1829) and phaC (slr1830) which encodesfor the PHB polymerase was deleted from the Synechocystis genome(through an intermediate strain Synechocystis SPA; Table 1) to obtainSynechocystis SPA:ΔphaEC strain. Next, three sets of 3HB pathway geneswere each introduced into this PHB polymerase-deletion strain in thesame way as described above, resulting in Synechocystis TESBd, HBd, TABdand TPUd. The genotypic purity of each strain was confirmed by colonyPCR in all cases.

Expression of 3HB Biosynthetic Genes in Synechocystis.

The 16S rRNA of both wild-type and engineered Synechocystis strains wasused as the reference to calculate the ΔC_(T) values for individualgenes when performing the RT-qPCR analysis. RT-qPCR analysis showed thatall target genes were successfully transcribed in all engineeredstrains, and that no detectable phaE or phaC expression was observed inany of the ΔphaEC strains (i.e., TESBd, TPUd, HBd, TABd) (Table 2). Inaddition, by introducing a Ptac promoter upstream of the nativephaA2-phaB2 operon, expression of phaA2 and phaB2 was enhanced in TPU3by nearly 6- and 120-fold, respectively. Similarly, expression of phaA2and phaB2 was enhanced by about 4- and 90-fold, respectively, in strainTPUd through the addition of Ptac.

Thus, it was found that the Ptac promoter could be used to effectivelytranscribe all 3HB pathway genes in Synechocystis 6803 and itsderivatives. Enzyme activity assay results (Table 3) revealed thatthiolase activities of the enhanced-expressed native PhaA2 and R.eutropha PhaA were 1.14±0.14 U (μmol/min/ml cell extract) and 13.12±3.36U, respectively. It is notable that the thiolase activity of PhaA wasabout 12-fold higher than that of PhaA2. In contrast, no thiolaseactivity was detected for the C. acetobutylicum Thil.(R)-3-hydroxybutyryl-CoA dehydrogenase activity was detected for PhaBwith a value of 0.23±0.15 U, but not for enhanced-expressed PhaB2.(S)-3-hydroxybutyryl-CoA dehydrogenase activity was negative in cellextract of strain HB5 (data not shown). The thioesterase activityreached a value of 0.484±0.044 U using decanoyl-CoA as substrate but6-fold lower, 0.084±0.021 U, when using butyryl-CoA as substrate (Table4), which was consistent with the report that TesB biases medium- andlong-chain fatty acyl-CoA substrates.

Production of 3HB by the Engineered Cyanobacteria.

After wild-type Synechocystis and eight engineered strains were grownphotosynthetically for five days, they all reached similar OD₇₃₀ of8.5-9.0 (FIG. 3). The culture broth was then sampled for HPLC analysis.Under the examined growth conditions, wild-type Synechocystis generatedand secreted into the culture medium up to 15.5 mg/l 3HB. Theintroduction and expression of tesB alone in Synechocystis (strain TESB)led to a final 3HB titer of about 20.6 mg/l. Interestingly, strain TPU3in which tesB as well as the native phaA2 and phaB2 were over-expressedwith the use of the Ptac promoter, achieved no detectable increase of3HB production compared to that of strain TESB. Co-expression of tesBwith thil and hbd of C. acetobutylicum (strain HB5) resulted in 33.2mg/l 3HB production. Production of 3HB was boosted to 45.1 mg/l in theculture medium of strain TAB1 which co-expressed tesB with phaA and phaBof R. eutropha. Deletion of phaE and phaC further improved 3HBproduction, as indicated by the ability of strain TABd to reach finaltiters of about 100 mg/l, nearly 6.5-fold higher than that of thewild-type (FIG. 4).

Notably, expression of E. coli tesB also resulted in a dramatic increaseof acetate production by our engineered Synechocystis relative to thewild-type (FIG. 4), consistent with the previous report about thetesB-over-expressed recombinant E. coli. Further experimental resultsindicated that TesB can catalyze the hydrolysis of acetyl-CoA to acetatewith a 33-fold lower activity than that in hydrolysis of butyryl-CoA(Table 5). Nevertheless, with expression of the R. eutropha phaA andphaB in strains TAB1 and TABd (Table 1), the acetate production wassignificantly reduced (FIG. 4). This is probably due to relatively highactivities of PhaA and PhaB which drove an increasing portion ofacetyl-CoA to form (R)-3-hydroxybutyryl-CoA (FIG. 1); the latter in turnoutcompeted acetyl-CoA as substrate for hydrolysis (by TesB), resultingin increase of 3HB production and decrease of acetate biosynthesis.

Improving 3HB Production by Nutrient Supplementation.

Studies found that nutrient starvation, specifically nitrogen orphosphate depletion, favors PHB accumulation in cyanobacteria. Thus,under these conditions, metabolic flux towards the common pathwayintermediate (R)-3-hydroxybutyryl-CoA (3HB pathway & PHB pathway;FIG. 1) should also be increased which might be leveraged to increase3HB production. We subjected Synechocystis TAB1 cells to nitrogenstarvation and normal BG11 medium cultures. The results showed thatnitrogen-starved cells were able to produce 3HB at higher titers thanthat of the control (TAB1 grown in BG11) during the first two days.However, 3HB production by the control then dramatically increased fromday 3, continuing on through day 6 (FIGS. 5A-C), resulting in overallhigher 3HB production than that of the nitrogen-starved counterparts(FIGS. 6A-C). Meanwhile, the cell density of nitrogen-starved culturestarted a gradual decline after day 2, whereas the cell density of thecontrol kept increasing until day 5 (FIGS. 6A-C). Once a little amountof nutrient was supplemented into the culture after day 7, both biomassand 3HB production started to increase again (FIG. 7).

From the above, it seemed low level of nutrient (herein most importantlynitrate) might positively support both 3HB production and cellviability, which would lead to increased 3HB production. As shown inFIGS. 8A-B, under all nutrient supplementation conditions, strain TABdachieved similar cell densities in about 5 days. The cell density of thenon-N and 5%-N supplementation cultures apparently started to decreaseafter day 7, which could be partially attributed to the daily samplingrather than severe collapse of the cell culture; in contrast, the 10%-Nsupplementation cultures were apparently able to maintain a relativelystable cell density after day 5.

Notably, after 10 days culture, chlorosis occurred in the non-N and 5%-Ncultures but not in the 10%-N culture (FIG. 9), suggesting pigmentedproteins had been degraded in the former cultures. Nevertheless, cellsfrom the yellow non-N and 5%-N cultures still maintained viability asthey could turn back to green the next day after being re-suspended infresh BG11 or BG11-10% N (containing merely 10% of the nitrate relativeto BG11) medium, respectively (FIG. 9). Under all three cultureconditions, 3HB production firstly underwent a 2-3 days lag phase andthen started to increase dramatically. The non-N and 5%-N culturescontinued this increase of 3HB titers until day 8 after which 5%-Ncultures exhibited a slight increase of 3HB titers while non-N culturesshowed a slight decrease of 3HB titers. In contrast, the 10%-N culturesexhibited a much lower 3HB production rate compare to that of 5%-N afterday 6. Eventually, The 3HB production achieved titers of 152.7±9.9 mg/lin the 10%-N cultures, 155.9±2.2 mg/l in the non-N cultures and191.0±10.3 mg/l in the 5%-N cultures (FIGS. 8A-B), indicating thatcompared to non-N and 10%-N supplementation strategy, 5%-Nsupplementation is more preferable for 3HB production under the examinedculture conditions.

3HB Production from Bicarbonate by Intermittent Medium Exchange.

Since 3HB can be secreted out of cells and our engineered Synechocystiscells still maintained viability after 10 days cultivation, we nextexamined the feasibility of applying engineered Synechocystis in acontinuous production mode where cells from the former 3HB productioncycle (Cycle I) can still be used for 3HB production in a latter cycle(Cycle II). We replaced the culture broth with fresh culture medium atthe end of each 10-day cultivation cycle for two reasons. First, dailysupplementation of NaHCO₃ would result in increasing Na⁺ ion in culturemedium which would cause salt stress and therefore impair the cellularactivity of cyanobacterial cells. Second, assimilation of HCO₃ ⁻(4H₂O+4HCO₃ ⁻→C₄H₈O₃+4OH⁻+9/2O₂) and NO₃ ⁻ (NO₃ ⁻+3H₂O→NH₄ ⁺+2OH⁻+2O₂)by Synechocystis would alkalize the culture medium and thus also causestress to cells. As a result, Synechocystis TABd exhibited durable andrepeatable activity in continuous production of 3HB under ourexperimental condition. The titers of 3HB in the culture medium couldachieve repeatable linear increase after a 2-3 days lag phase at thebeginning of each cycle and could finally reach 3HB titers of 191.0±10.3mg/l (for Cycle I) and 203.3±10.1 mg/l (for Cycle II), respectively(FIGS. 10A-B). Carbonate also is believed to be usable for the carbonsource.

3HB Production from Atmospheric CO₂.

The ability of Synechocystis strain TABd to photosynthetically produce3HB using CO₂ as sole carbon source was then investigated by continuousaeration of cultures with ambient air. Upon overcoming a lag phase ofnearly one week (during which significant biomass growth was observed),3HB production by Synechocystis TABd then quickly accelerated, achievinga titer of 446.5±31.0 mg/l after 18 days of continuous cultivation. Atthis point, daily BG11 addition into the culture was arrested from days19 through 21 to probe its effect on continued 3HB production. As can beseen in FIG. 11, 3HB titers continued to increase regardless, andreached a final titer of 533.4±5.5 mg/l by the end of day 21.

It should be noted that at this point, there was no indication that 3HBproduction would stop; however, we merely elected to stop theexperiment. From FIG. 11, it was also observed that starting from day 7,as cell growth declined 3HB production rates were found to dramaticallyincrease (FIG. 7). The relationship here between biomass growth and 3HBproduction rate is consistent with the results of the former experimentsin which NaHCO₃ was used as the sole carbon source (FIGS. 8A-B, 6A-C and7).

The above observations pointed to the possibility of using engineeredSynechocystis for continuous 3HB production. By following a mediumexchange protocol, stable and continuous 3HB production was maintainedfor a total of 20 days (i.e., two 10-day cycles), resulting in final 3HBtiters of ˜200 mg/l at the end of each cycle (FIGS. 10A-B). Theexperiments were stopped after 10 days cultivation due to increasingstresses such as increasing salt concentration and increasing pH in theculture broth. A longer period 3HB production process was developed byusing atmospheric CO₂ rather than bicarbonate as carbon source, and theresults showed that a titer of 533.4±5.5 mg/l 3HB in the culture brothwas achieved after 21 days cultivation when cells still kept viability(FIG. 11).

Both of the experiments above demonstrated that by being cultivated andimmobilized in a properly controlled photo-bioreactor system, ourengineered Synechocystis strains could be employed into a continuousprocess for 3HB production using only CO₂, water and low-cost inorganiccompounds as feed stocks and sun light as the energy source. We areexpecting that such a carbon-neutral and sustainable process wouldsignificantly decrease the manufacture cost in production of 3HB as wellas other useful chemicals that can be expanded to.

TABLE 1 Strains and plasmids used in this disclosure Genotype* ReferenceStrains E. coli XL 1-Blue Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44thi-1 recA1 gyrA96 Stratagene MRF′ relA1 lac [F′ proAB lacI^(q)ZΔM15Tn10 (Tet^(r))] B. subtilis 168 ATCC Synechocystis PCC6803 Wild-typeATCC TESB Ptac-tesB-Kan^(R) integrated at S2 site in Synechocystis 6803This study TPU3 Ptac-tesB-Kan^(R) integrated at S2 site and Cm^(R)-Ptacintegrated at S4 site This study HB5 Ptac-tesB-Kan^(R) integrated at S2site and Cm^(R)-Ptac-thil-hbd integrated at S1 This study site TAB1Ptac-tesB-Kan^(R) integrated at S2 site and Cm^(R)-Ptac-phaA-phaBintegrated at This study S1 site SPA Ptac-adhe2 integrated at S2 siteStored in lab SPA:SPSK3 Ptac-adhe2 integrated at S2 site andPtac-sacB-Kan^(R) integrated at S3 site This study SPA:ΔphaEC Ptac-adhe2integrated at S2 site, phaE and phaC deleted at S3 site This study TESBdphaE and phaC deleted at S3 site, Ptac-tesB-Kan^(R) integrated at S2site This study TPUd phaE and phaC deleted at S3 site, Ptac-tesB-Kan^(R)integrated at S2 site, Cm^(R)- This study Ptac integrated at S4 site HBdphaE and phaC deleted at S3 site, Ptac-tesB-Kan^(R) integrated at S2site, Cm^(R)- This study Ptac-thil-hbd integrated at S4 site TABd phaEand phaC deleted at S3 site, Ptac-tesB-Kan^(R) integrated at S2 site,Cm^(R)- This study Ptac-phaA-phaB integrated at S4 site PlasmidspBluescript II Amp^(R), pUC ori, f1(+) ori Stratagene SK(+) pACYC184Cm^(R), Tet^(R), p15A ori New England BioLabs pET-30a(+) Kan^(R), lacI,pBR322 ori, f1 ori Novagen pETphaAphaB phaA, phaB integrated between theNcoI and AvrII sites of pETDuet-1 Nielsen's (Amp^(R), pBR322 ori) LabpBS-SRSL SR12-SL12 inserted between the SacI and KpnI sites ofpBluescript II SK(+) This study pBS-SPT Ptac-thil integrated between theBamHI and SalI sites of pBS-SRSL This study pBS-SPTH hbd integratedbetween the NcoI and SalI sites of pBS-SPT This study pBS-SCatNcoI-removed cat (Cm^(R)) integrated into the PstI and BamHI sites ofpBS- This study SRSL pBS-SCPTH Ptac-thil-hbd integrated between the NcoIand SalI sites of pBS-SPT This study pBS-SCPTH2 MluI site added betweenthil and hbd of pBS-SCPTH pBS-SCPTB phaB inserted between the MluI andHindIII sites of pBS-SCPTH2 This study pBS-SCPAB Ptac-phaA insertedbetween the BamHI and MluI sites of pBS-SCPTB This study pBS-SCG GTPfragment inserted between the SacI and PstI sites of pBS-SCat This studypBS-GCPU Ptac-PHAU inserted between the BamHI and KpnI sites of pBS-SCGThis study pBS-S2 SR56-SL56 inserted into the SacI and XhoI sites ofpBluescript II SK(+) This study pBS-S2K kan (Kan^(R)) inserted betweenMluI and SalI sites of pBS-S2 This study pBS-SPtTeK Ptac-tesB integratedbetween BglII and HindIII site of pBS-S2K This study pBS-SPTK PpsaD-thilintegrated between the BamHI and MluI sites of pBS-S2K This studypBS-SPtK Ptac inserted between the BamHI and NdeI sites of pBS-SPTK Thisstudy pBS-SPSK2 sacB inserted between the NdeI and MluI sites of thepBS-SPtK This study pBS-PHA PHA inserted between the XhoI and SacI sitesof pBS-S2 This study pBS-SPSK3 Ptac-sacB-kan of pBS-SPSK2 insertedbetween BamHI and SalI of pBS-PHA This study *S1, the site on the genomeof Synechocystis 6803 between slr1495 and sll1397; S2, the site betweenslr1362 and sll1274; S3, the site between slr1828 and sll1736; S4, thesite between slr1992 and phaA2.

TABLE 2 Expression analysis of 3HB pathway genes by RT-qPCR* ΔC_(T) GeneWT TPU3 HB5 TAB1 TESBd TPUd HBd TABd tesB n.d. 10.87 ± 0.07 10.37 ± 0.2211.19 ± 0.11 10.44 ± 0.12 12.05 ± 0.24 10.83 ± 0.15 10.94 ± 0.12 phaA220.85 ± 0.06 18.17 ± 0.16 — — — 18.75 ± 0.26 — — phaA n.d. — — 12.24 ±0.01 — — — 12.04 ± 0.11 thil n.d. — 15.28 ± 0.09 — — — 15.30 ± 0.46 —phaB2 19.30 ± 1.07 12.42 ± 0.28 — — — 12.84 ± 0.07 — — phaB n.d. — —12.25 ± 0.26 — — — 12.50 ± 0.05 hbd n.d. — 17.14 ± 0.26 — — — 17.62 ±0.01 — phaE 15.52 ± 0.28 — — 15.76 ± 0.26 n.d. n.d. n.d. n.d. phaC 17.17± 0.48 — — 17.20 ± 0.30 n.d. n.d. n.d. n.d. *“n.d.”, “not detectable”.“—”, experimental data was not available. The relative abundance ofdifferent mRNA molecules could be estimated using 2^(−ΔC) _(T); thebigger the ΔC_(T) value, the lower abundance of the corresponding mRNAis.

TABLE 3 Enzyme activities for engineered strains. Strains with sameExamined expression Enzyme (gene) Activities* strain cassette Thiolase(phaA2) 1.14 ± 0.14 TPU3 TPUd Thiolase (thil) n.d. HB5 HBd Thiolase(phaA) 13.12 ± 3.36  TAB1 TABd (R)-3-Hydroxybutyryl-CoA n.d. TPU3 TPUddehydrogenase (phaB2) (S)-3-Hydroxybutyryl-CoA n.d. HB5 HBddehydrogenase (hbd) (R)-3-Hydroxybutyryl-CoA 0.23 ± 0.15 TAB1 TABddehydrogenase (phaB) Thioesterase (tesB) 0.084 ± 0.021 TESB TPU3, HB5,TAB1, TESBd, TPUd, HBd, TABd *Enzyme activities were given inμmol/min/mL cell extract; “n.d.” stands for “not detectable”. “—” meansexperimental data was not available.

TABLE 4 Thioesterase (TesB) Substrate activities* Examined strainDecanoyl-CoA 0.484 ± 0.044 Synechocystis strain TESB Bulyryl-CoA 0.084 ±0.021 Synechocystis strain TESB Acetyl-CoA n.d. Synechocystis strainTESB

TABLE 5 Thioesterase (TesB) Substrate activities* Examined strainButyryl-CoA 10.451 ± 1.924 E. coli XL 1-Blue/pBS-SPtTeK Acetyl-CoA 0.319 ± 0.022 E. coli XL 1-Blue/pBS-SPtTeK Butyryl-CoA n.d. E. coli XL1-Blue/pBS-S2K Acetyl-CoA n.d. E. coli XL 1-Blue/pBS-S2K *Enzymeactivities were given in μmol/min/mL cell extract; “n.d.” stands for“not detectable”.

TABLE 6 Strains and plasmids used in this paper. Strains Genotype*References E. coli Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1Stratagene XL1-Blue recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn10(Tet^(r))] MRF′ Synechocystis ABd P_(tac)-adhe2 integrated at S2 site,phaE and phaC deleted at S3 This study site, Cm^(R)-P_(tac)-phaA-phaB1integrated at S1 site TTrK P_(tac)-tesB-T1-Kan^(R) integrated at S2site, Cm^(R)-P_(tac)-phaA- This study phaB1 integrated at S1 site, phaEand phaC deleted at S3 site SD-TrK P_(tac)-SD-tesB-T1-Kan^(R) integratedat S2 site, Cm^(R)-P_(tac)-phaA- This study phaB1 integrated at S1 site,phaE and phaC deleted at S3 site UTR-TrK P_(tac)-UTR-tesB-T1-Kan^(R)integrated at S2 site, Cm^(R)-P_(tac)-phaA- This study phaB1 integratedat S1 site, phaE and phaC deleted at S3 site PTrK12P_(psbA12)-tesB-T1-Kan^(R) integrated at S2 site, Cm^(R)-P_(tac)-phaA-This study phaB1 integrated at S1 site, phaE and phaC deleted at S3 sitePTrK14 P_(psbA14)-tesB-T1-Kan^(R) integrated at S2 site,Cm^(R)-P_(tac)-phaA- This study phaB1 integrated at S1 site, phaE andphaC deleted at S3 site PTrK16 P_(psbA12)-P_(tac)-tesB-T1-Kan^(R)integrated at S2 site, Cm^(R)-P_(tac)- This study phaA-phaB1 integratedat S1 site, phaE and phaC deleted at S3 site SPA:ΔphaEC P_(tac)-adhe2integrated at S2 site, phaE and phaC deleted at S3 This study siteABd-SPSK2 P_(tac)-sacB-kan integrated at S2 site, phaE and phaC deletedat This study S3 site, Cm^(R)-P_(tac)-phaA-phaB1 integrated at S1 siteABd-TTe Ptac-tesB integrated at S2 site, phaE and phaC deleted at S3This study site, Cm^(R)-P_(tac)-phaA-phaB1 integrated at S1 site TTB2K3phaE and phaC deleted at S3 site, P_(tac)-tesB integrated at S2 Thisstudy site, P_(tac)-tesB_(opt)-phaB2_(eu-opt)-kan integrated at S3 site,Cm^(R)- P_(tac)-phaA-phaB1 integrated at S1 site ABdTB phaE and phaCdeleted at S3 site, P_(tac)-tesB_(opt)-phaB2_(eu-opt) This studyintegrated at S3 site, Cm^(R)-P_(tac)-phaA-phaB1 integrated at S1 sitePTrK16-Int P_(psbA12)-P_(tac)-tesB-T1-Kan^(R) integrated at S2 site,phaE and This study phaC deleted at S3 site R154P_(psbA12)-P_(tac)-tesB-T1-Kan^(R) integrated at S2 site, phaE and Thisstudy phaC deleted at S3 site, Cm^(R)-P_(tac)-phaA-(RBS_(opt))-phaB1integrated at S1 site R168 P_(psbA12)-P_(tac)-tesB-T1-Kan^(R) integratedat S2 site, phaE and This study phaC deleted at S3 site,Cm^(R)-P_(psbA12)-P_(tac)-phaA-(RBS_(opt))- phaB1 integrated at S1 site*S1, the site on the genome of Synechocystis 6803 between slr1495 andsll1397; S2, the site between slr1362 and sll1274; S3, the site betweenslr1828 and sll1736.

TABLE 7 Enzyme activity assay for engineered Synechocystis strains*.Acetoacetyl- CoA Strain Genotype reductase Thioesterase TTrKP_(tac)-tesB, P_(tac)-phaA-phaB1, 0.063 ± 0.013 0.345 ± 0.010 ΔphaECPTrK16 P_(psbA12)-P_(tac)-tesB, P_(tac)- 0.078 ± 0.033 0.243 ± 0.014phaA-phaB1, ΔphaEC TTB2K3 P_(tac)-tesB, P_(tac)-phaA-phaB1, 0.093 ±0.007 0.386 ± 0.010 P_(tac)-tesB_(opt)-phaB2_(eu-opt), ΔphaEC ABdTBP_(tac)-phaA-phaB1, P_(tac)- n.a. 0.161 ± 0.014tesB_(opt)-phaB2_(eu-opt), ΔphaEC R154 P_(tac)-tesB, P_(tac)-phaA- 0.139± 0.020 n.a. (RBS_(opt))-phaB1, ΔphaEC *The cells for enzyme assay werecollected after growing in BG11 (10 mM TES-NaOH, pH 8.0) for 12 hoursunder an irradiation of 60 μE/m²/s.

Further Examples Materials and Methods Culture Conditions

All recombinant plasmids were constructed and stored using E. coliXL1-Blue MRF′ (Stratagene, La Jolla, Calif.) as the host strain.Synechocystis strains were grown in BG11 medium (Rippka et al., 1979)supplemented with 50 mM NaHCO₃ under a light intensity of 60 μE/m²/sunless otherwise specified. For BG11-agar plates, 10 mM TES (pH 8.0), 3g/L thiosulfate and 1.5% agar was supplemented into BG11 medium beforeautoclaving.

Modification of Synechocystis Genome

The chromosome of Synechocystis 6803 was modified using the same methodsas described previously (Wang et al., 2013). The genotype of eachengineered Synechocystis strain is described in Table 6. The genotypicpurity of each strain was achieved by a series of streaking of thecolonies on the antibiotic-supplemented BG11 plates and was confirmed bycolony PCR.

Production of (R)-3HB from Bicarbonate

Synechocystis strains were inoculated in 50 ml flasks containing 10 mlBG11 (10 mM TES-NaOH) to an initial OD₇₃₀ of 2.0. Then, cells wereincubated in a shaking bed (150 rpm) at 30° C. with a light intensity of60 μE/m²/s except otherwise specified. Every day, 0.05 ml cell culturewas sampled for analysis of the OD₇₃₀ before 0.5 ml 1.0M NaHCO₃ wasadded to each culture and the pH was adjusted to ˜8.0 by 10 N HCl. Eachcell culture was sampled at the end of day 3 and day 5, respectively,for analysis of the (R)-3HB titers. All culture experiments wereconducted at least in triplicate for each strain.

Production of 3HB from Carbon Dioxide

Synechocystis was inoculated into a 125 ml flask containing 50 mlautoclaved BG11 (10 mM TES-NaOH) medium to an initial OD₇₃₀ of 0.2. Theculture was placed at 30° C. with continuous illumination of 100μE/m²/s, bubbled with ambient air during the first 24 h and thenswitched to 1% (v/v) CO₂. The aeration rate was set as 37.5 mL/min.Every day, 1 mL of culture was sampled and 1.25 mL 2-fold concentratedsterilized BG11 medium was added back into the culture until day 21. Theexperiments were conducted in duplicates.

Gene Expression Analysis by RT-qPCR

Cells were resuspended to an initial OD₇₃₀ of 2.0 before they were grownin BG11 (10 mM TES-NaOH) medium under continuous illumination of 60μE/m²/s. Daily, 0.05 mL cell culture was sampled for analysis of theOD₇₃₀ before 0.5 mL 1.0M NaHCO₃ was added to each culture and the pH wasadjusted to ˜8.0 by 10 N HCl. At 3.5 days of cultivation, approximately1.67×10⁸ Synechocystis cells (assuming OD₇₃₀ of 0.6 equals to 10⁸cells/ml; Liu et al., 2011) were collected by centrifugation at 17,000g, 4° C. for 1 min. The supernatant was discarded and the cell pelletwas used for RNA extraction using ZR Fungal/Bacterial RNA MiniPrep™ Kit(ZYMO Research, Irvine, Calif.). The RNA was then quantified by RT-qPCRusing methods described previously (Gao et al., 2011). The primers usedfor RT-qPCR analysis is listed in Supplementary Data.

Enzyme Activity Assay

Synechocystis cells were grown as described above for 12 hours. At theend of the cultivation, approximately 1.67×10⁹ Synechocystis cells werecollected by centrifugation at 8000 g, 4° C. for 5 min. The supernatantwas discarded and the cell pellets were frozen on dry ice and stored at−80° C. before the assay. For the thioesterase enzyme activity assay,the cell pellet was resuspended with 500 μL ice-cold 0.1 M Tris-HCl (pH7.5) and lysed by sonication (100 cycles of 3-s-on/3-s-off) in ice bath.The cell lysate was centrifuged at 17000 g, 4° C. for 10 min before thesupernatant was analyzed for the thioesterase activity following theprevious protocols but using Butyryl-CoA as the substrate (Wang et al.,2013). For the acetoacetyl-CoA reductase enzyme activity assay, the cellpellet was resuspended in 500 μL ice-cold Buffer A [50 mM K₂HPO₄—HCl (pH7.5), 10% glycerol, 1 mM EDTA, 1 mM DTT] with 0.1 mM PMSF and lysed bysonication (20 cycles of 3-s-on/3-s-off) in ice bath. The supernatantwas analyzed for the acetoacetyl-CoA reductase activity using theprotocol established previously (Wang et al., 2013).

Product Quantification

The (R)-3HB and acetate concentrations were quantified by an 1100 seriesHPLC using the method described previously (Wang et al., 2013). Briefly,samples of the Synechocystis culture were centrifuged at 17,000 g for1˜2 min at room temperature and the supernatant was properly dilutedbefore being analyzed on HPLC equipped with an Aminex HPX-87Hanion-exchange column (Bio-Rad Laboratories, Hercules, Calif.) and arefractive index detector (Agilent, Santa Clara, Calif.). The columntemperature was maintained at 35° C. during operation. The mobile phasewas 5 mM H₂SO₄ and the flow rate was set as a linear gradient from 0.55ml/min to 0.8 ml/min over 12 min, followed by an 8 min hold (Tseng etal., 2009).

Results and Discussion Construction of a Promoter Library

Since promoter is the key element to initiate the expression of theinterest genes, to screen out a strong promoter is critical to improvingthe gene expression level in the host strain. Herein, a promoter librarywas constructed for screening promoters with desirable performance. Thelibrary included the constitutive P_(tac) promoter, the wild typelight-inducible P_(psbA2) promoter from Synechocystis 6803, and threederivative promoters (FIG. 13). Synechocystis strain TTrK, with all the3HB biosynthesis genes expressed via the Ptac promoter, wasreconstructed from the TABd strain (Wang et al., 2013) by placing anadditional rrnB T1 terminator downstream the tesB gene (Table 6). TheP_(tac) promoter upstream the tesB gene was replaced by the wild typepsbA2 promoter (thereafter P_(psbA12)), resulting in strain PTrK12. Itwas reported that the 5′-untranslated region (UTR) of the P_(psbA12)promoter from Synechocystis 6803 plays an important role in stabilizingthe psbA2 mRNA (Sakurai et al., 2012). It was therefore assumed that the5′-UTR of P_(psbA12) promoter might be placed upstream of the interestgene to increase the mRNA stability of the corresponding downstreamgene(s). To this end, the whole 5′-UTR or merely the ribosome bindingsite (RBS, including the SD sequence) of the psbA2 gene was placeddownstream of the P_(tac) promoter, resulting in promoters P_(tac)-UTRor P_(tac)-SD, respectively (FIG. 13). In addition, since the AU-box inthe 5′-UTR of P_(psbA2) was suggested to be a negative element for geneexpression (Agrawal et al., 2001; Sakurai et al., 2012), the AU-box hasbeen deleted from the P_(psbA12) promoter to form promoter P_(psbA14)(FIG. 13).

The Performance of Promoters

Since activities of the thioesterase (encoded by tesB) and theacetoacetyl-CoA reductase (encoded by phaB1) were identified as thepossible bottlenecks for (R)-3HB biosynthesis in Synechocystis based onthe enzyme activity assay results (Wang et al., 2013, 2014), theperformance of the above five promoters were characterized throughexpressing the tesB gene in the phaA-phaB-expressing strains (Table 6).When the engineered strains were grown under photoautotrophicconditions, no significant difference was observed among the strainsregarding the cell growth rates (FIG. 14A-D). However, the 3HBproduction titers exhibited considerable variability among the fivestrains (FIG. 14B). Strain TTrK (Pac) exhibited the highest 3HBproductivity, reaching 176.9±6.4 mg/L in five days. Strain PTrK12(P_(psbA12)) produced 166.1±5.2 mg/L of 3HB, slightly lower than that ofstrain TTrK (P_(tac)). The 3HB productivities of strains UTR-TrK(P_(tac)-UTR) and PTrK14 (P_(psbA14)) were slightly lower than that ofstrain PTrK12 (P_(psbA12)), reaching 147.5±13.3 mg/L and 141.3±7.3 mg/L,respectively. Strain SD-TrK (P_(tac)-SD) produced the least amount of3HB, reaching only 70.2±7.0 mg/L, less than half of that of any otherstrain (FIG. 14B).

RNA analysis of the above five strains indicated that the mRNA level ofgene tesB in strain TTrK was the highest among all investigated strains(FIG. 14C) which is consistent with its highest 3HB productivity (FIG.14B), suggesting that the P_(tac) promoter might be the strongestpromoter under the examined culture condition. In contrast, the tesBmRNA level in strain SD-TrK was the lowest (FIG. 14C). Sincemodification of the 5′-UTR was assumed to have little impact on the genetranscription, our finding indicated that the apparent lower abundanceof the tesB mRNA in SD-TrK was attributed to the poorer stability of themRNA product that contained the RBS region of the P_(psbA12) promoter.This result was consistent with the previous report that the RBS of theP_(psbA12) promoter was a target of the RNase E/G in Synechocystis 6803(Horie et al., 2007; Sakurai et al., 2012). Interestingly, the tesB mRNAabundance was 57% higher in the strain UTR-TrK compared to that ofstrain SD-TrK, consistent with the ˜2-fold higher 3HB production rate(FIG. 14B). It suggested that the AU-box and the 19-bp upstream sequence(from the P_(psbA12) promoter) in the P_(tac)-UTR promoter has apositive impact on expression of the downstream gene. While the AU-boxis also a target of the RNase E, this region may be bound by theintrinsic cis-coded as RNA “PsbA2R” and therefore protect the mRNA fromdegradation by endoribonuclease RNase E/G in Synechocystis (Horie etal., 2007; Sakurai et al., 2012). Moreover, after the AU-box wasremoved, the P_(psbA14) promoter did not show any improved expression ofthe tesB mRNA (FIG. 14C) and in contrast, the 3HB and acetate productiontiters slightly decreased compared to those of the strain PTrK12 (FIG.14B, D).

Interestingly, although the tesB expression levels in strains SD-TrK,UTR-TrK, PTrK12 and PTrK14 range from 30% to 60% relevant to that of thestrain TTrK (FIG. 14C), the acetate accumulation in these strainsreached merely less than 20% of that of strain TTrK (FIG. 14D). TesB haslow activity on acetyl-CoA (Wang et al., 2013), and the acetyl-CoA poolin each strain is presumably same as indicated by the same cell growthrate (FIG. 14A). Therefore, the acetate titer may serve as an indicatorof the TesB enzyme activity in each strain. The unmatched TesB enzymeactivities (FIG. 14C) and mRNA abundance (FIG. 14D) is probablyattributed to the different translation efficiency of differentpromoters, which is discussed thoroughly later on.

Construction and Characterization of a Dual Promoter

The P_(tac) and the P_(psbA12) promoter were recombined together to forma cascade structure (FIG. 15A) in order to investigate if the expressionof the downstream gene can be enhanced in such a way. As a result, therewas no significant difference between strains PTrK16 and TTrK regardingto the cell growth rate (FIG. 15C), (R)-3HB production and acetateaccumulation (FIG. 15C). RT-qPCR analysis results indicated that theabundance of tesB mRNA in strain PTrK16 and TTrK was at the same levelon day 3.5 (FIG. 15D), though the tesB mRNA in strain PTrK16 reachedonly approximately 70% of that in strain TTrK on day 0.5. The resultswere further confirmed by the enzyme activity assay (FIG. 15E; Table 7).

Mitigation of the Rate-Limiting Step in (R)-3HB Biosynthesis withIncreased Light Intensity

Though the tesB mRNA abundance was 2-fold higher (FIG. 14C) and thethioesterase activity (indicated by the acetate titer; FIG. 14D) wasmore than 5-fold higher in strain TTrK compared to that of strainUTR-TrK, it merely led to marginal increase in production of (R)-3HB instrain TTrK (FIG. 14B). It implied that the metabolic flux between theacetyl-CoA and the (R)-3HB-CoA might be the rate-limiting factor for(R)-3HB biosynthesis (FIG. 12). In other words, the thiolase (PhaA) orthe acetoacetyl-CoA reductase (PhaB) activity might be the rate-limitingstep for (R)-3HB biosynthesis. Thiolase PhaA catalyzes the condensationof two molecules of acetyl-CoA to produce acetoacetyl-CoA, but thisreaction is reversible and it strongly favors the hydrolysis ofacetoacetyl-CoA. The k_(cat)[condensation] is 11-fold smaller thank_(cat)[hydrolysis], whereas the K_(m) on acetyl-CoA is 78-fold higherthan the K_(m) on acetoacetyl-CoA (Masamune et al., 1989). The activityof thiolase PhaA on hydrolyzing acetoacetyl-CoA was previously measuredto be approximately 200-fold higher than the activity of theacetoacetyl-CoA reductase PhaB on synthetically reducing acetoacetyl-CoAto form (R)-3HB-CoA (Wang et al., 2013, 2014). These findings suggeststhat the activity of the acetoacetyl-CoA reductase PhaB on syntheticallydrawing off acetoacetyl-CoA was probably the bottleneck for (R)-3HBbiosynthesis.

Increasing the co-factor availability has been proven as an effectiveapproach to improving the enzyme activity (Niederholtmeyer et al., 2010;Shen et al., 2011). NADPH, product of the photosynthesis process, is notonly the driving force for carbon fixation in the Calvin cycle, but alsothe co-factor for the acetoacetyl-CoA reductase (PhaB1) in the (R)-3HBbiosynthesis pathway (FIG. 12). Since photosynthesis activity ispositively associated with the photon flux at lower range of lightintensity (<200 μE/m²/s) (Williams and Laurensa, 2010; Yang et al.,2011), the next step was to cultivate strains PTrK12 and TTrK underdifferent intensity of light in hope of stimulating the (R)-3HBproduction via optimizing the photosynthesis activity and theintracellular NADPH pool.

For strain TTrK (or TABd) when the light intensity was increased from 35μE/m²/s to 60 μE/m²/s, the (R)-3HB production was elevated from 93.9mg/L to 176.4 mg/L, nearly 2-fold increase. However, when the lightintensity was further increased from 60 μE/m²/s to 150 jE/m²/s, noobvious improvement was observed in regard to the (R)-3HB productivity(FIG. 16A). In contrast, the acetate titer was increased from 16.6 mg/Lto 36.5 mg/L to 78.6 mg/L when the light intensity was increased from 35μE/m²/s to 60 μE/m²/s to 150 μE/m²/s (FIG. 16A). For strain PTrK12, the(R)-3HB productivity was increased by ˜20% when the light intensity wasincreased from 60 μE/m²/s to 150 μE/m²/s (FIG. 16B), whereas the acetatetiter was elevated to 3-fold higher, reaching 21.6 mg/L (FIG. 16B). Overall, it was found that the intracellular acetyl-CoA pool was graduallyelevated with the increase of the light intensity from 35 μE/m²/s to 150μE/m²/s which indicated increased activity of the photosynthesis, carbonfixation and probably also the intracellular NADPH abundance (FIG. 12),consistent with the literature (Williams and Laurensa, 2010; Yang etal., 2011). However, the (R)-3HB production ceased increasing withincreased illumination when the light intensity surpassed 60 μE/m²/s.Based on these results, it was surmised that the expression of theacetoacetyl-CoA reductase (PhaB1) rather than supply of the co-factorNADPH was probably the rate-limiting factor for (R)-3HB production.

Multiple Copies of phaB and tesB

In order to increase the acetoacetyl-CoA reductase activity, gene phaB2of R. eutropha H16 that encodes an isozyme of PhaB1 was de novosynthesized after codon optimization (phaB2_(eu-opt)), placed under thecontrol of the P_(tac) promoter and inserted into the chromosome ofSynechocystis ABd-TTe [P_(tac)-phaA-phaB1, P_(tac)-tesB, ΔphaEC]. Duringthe genetic manipulation, the tesB gene was also codon-optimized, denovo synthesized (tesB_(opt)) and placed downstream of thephaB2_(eu-opt) gene before being integrated into the chromosome of theSynechocystis ABd-TTe. The resultant strain was denominated asSynechocystis TTB2K3 [P_(tac)-phaA-phaB1, P_(tac)-tesB,P_(tac)-tesB_(opt)-phaB2_(eu-opt), ΔphaEC] (Table 6). Strain TTB2K3exhibited the same growth rate under illumination of 60 μE/m²/s comparedto that of strain TTrK (FIG. 17A). However, the TTB2K3 strain was ableto produce (R)-3HB to a titer of 285.1 mg/L after five days ofcultivation, which was 1.6-fold higher than that of strain TTrK (FIG.17B). It was also noteworthy that the acetate production by strainTTB2K3 was increased to 74.4±4.3 mg/L (vs. 41.3±4.3 mg/L for strainTTrK), probably due to the increased thioesterase activity (Table 7).

One copy of the tesB gene was then removed from the chromosome of strainTTB2K3 to construct strain ABdTB (Table 6) in order to decrease theacetate production as well as to verify if the thioesterase activity wasthe rate-limiting step for (R)-3HB biosynthesis. It was found that whilethe acetate production was decreased from 74.4 mg/L to 62.8 mg/Lprobably due to the decreased thioesterase activity (Table 7), strainABdTB exhibited similar growth rate and (R)-3HB productivity compared tothat of strain TTB2K3 (FIG. 17). Based on these results, we confirmedthat the rate-limiting factor for (R)-3HB production in aboveSynechocystis strains was not the thioesterase (TesB) activity butinstead was the acetoacetyl-CoA reductase (PhaB) activity.

Optimization of the Ribosome Binding Site for phaB1

Since the ribosome binding site (RBS) plays a crucial role in initiatingthe translation of the corresponding gene, the RBS for genes of interestneed to be optimized in order to enhance the expression of the (R)-3HBbiosynthesis genes. Previously, it was recognized that theShine-Dalgarno (SD) sequence UAAGGAGG, which is perfectly complementaryto the 3′-terminal sequence of the 16S rRNA in Escherichia coli K12strain could enable 3- to 6-fold higher translation efficiency than theSD sequence AAGGA, regardless of the spacing between the SD and thetranslation start codon—ATG (Makrides, 1996). In this study, the RBS wasexamined upstream of each open reading frame of the 3HB biosynthesisrelevant genes. It was found that the SD sequence upstream of the genephaB1, AAGGAGTGG, was not a perfect match to the 3′-terminal sequence(5′-ACCUCCUUU-3′) of the 16S rRNA in Synechocystis 6803 (Wang et al.,2012). The original SD sequence for phaB1 was therefore replaced bysequence AAGGAGGT (RBS_(opt)) which is fully complementary with the3′-terminal sequence of 16S rRNA of Synechocystis 6803 (FIG. 18A). Theresultant strain with the RBS_(opt) for gene phaB1 was denominated asR154 (Table 6).

It was found that the acetoacetyl-CoA reductase (PhaB) activity wasincreased by 2.2-fold in strain R154 compared to that of strain TTrK(FIG. 18B). While the growth of strain R154 exhibited a similar patterncompared to strain TTrK (FIG. 18C), strain R154 was able to produce(R)-3HB at a titer that was 1.6-fold higher than that of strain TTrK,reaching 280.2 mg/L after five days of cultivation (FIG. 18D). Itsuggested that the new RBS_(opt) was much more efficient in initiatingthe translation of the gene phaB1 compared to the original RBS, and RBSoptimization for genes of interest is an effective approach to enhancingthe chemical production in Synechocystis. Further replacing the P_(tac)promoter with the dual promoter P_(psbA12)-P_(tac) for the phaA-phaB1operon in Synechocystis strain R168 (Table 6) resulted in littledifference in regard to the cell growth rate and the production of(R)-3HB and acetate (data not shown).

Enhanced Production of (R)-3HB from CO₂

The ability of Synechocystis strains to photosynthetically produce(R)-3HB directly from CO₂ was then examined by continuously aeratingcultures with 1% CO₂. As shown in FIG. 19A, Synechocystis strains R168and ABdTB underwent relatively fast growth during the first four to fivedays, and the growth rate slowed down thereafter. In contrast, the(R)-3HB production quickly accelerated after the first three to fourdays (FIG. 19B, FIG. 9), consistent with our previous observation (Wanget al., 2013). Starting from day 4 until day 21 (when the experiment waselectively stopped), strains R168 and ABdTB exhibited average (R)-3HBproduction rates of ˜86 and ˜65 mg/L/day, respectively, with peakproductivities of 208 and 139 mg/L/day, respectively. Eventually,(R)-3HB was able to accumulate in the culture medium to titers of 1347and 987 mg/L, respectively, after 21 days of continuous cultivation(FIG. 19B). It is noteworthy that the cumulative titer of (R)-3HB wasable to reach 1599 mg/L for strain R168 at the end of the cultivation(FIG. 20).

The dramatic increase of the (R)-3HB production rate compared to theprevious result (Wang et al., 2013) could probably be attributed to thefollowing reasons. First, the enzyme activity of acetoacetyl-CoAreductase, which is the bottleneck in the (R)-3HB biosynthesis pathwayidentified in this study (FIG. 17B, 18C), was increased by 1.5- and2.2-fold in strains ABdTB and R154, respectively (Table 7), leading todramatic increase of the metabolic flux towards the (R)-3HBbiosynthesis. Second, as photoautotrophic growth of Synechocystisalkalizes the medium (Summerfield and Sherman, 2008), the pH of theculture medium increased to 10˜11 when ambient air (with 0.04% CO₂) wasaerated into the culture in the previous study (Wang et al., 2013),indicating that the CO₂ supply was not able to meet the demand ofSynechocystis cells. In this study, with aerating 1% CO₂ into theculture, the pH of the culture medium was able to be maintained at ˜8.0during the whole cultivation process, indicating that the CO₂ supply wassufficient in this study. Third, in contrast to cultivation ofSynechocystis cells by simply bubbling air into the flasks placed in astatic incubator as described in the previous study (Wang et al., 2013),in this study the flasks containing Synechocystis cells were placed in ashaker with a rotation rate of 150 rpm during aeration, which resultedin more even distribution of the supplied CO₂ and photons in the cellpopulation.

CONCLUSION

In order for cyanobacterial biotechnology to be economically feasible,chemicals of interest need to be produced at desirable high titers sothat the expense in purification of unit amount of the product can besignificantly reduced. To date, however, it remains a challenge toconstruct such a type of high-productivity cyanobacterial strains.Typically, the titers of chemicals that are photosynthetically producedby engineered cyanobacteria are below 1 g/L (Wang et al., 2012). Thesituation is partially due to limited well-characterized genetic toolsand low level expression of the genes of interest.

In this study, a total of six promoters were completely characterized inSynechocystis and it was found that the constitutive Pac promoter wasthe strongest in expressing the target gene under the examinedexperimental conditions (FIG. 14C). P_(tac) and the light-inducibleP_(psbA12) promoter exhibited the best performance in regard tophotosynthetic production of (R)-3HB (FIG. 14B). Recombination of theP_(psbA12) and P_(tac) to form a dual promoter resulted in approximatelythe same gene expression efficiency compared to that of the P_(tac)promoter alone (FIG. 15C-E). The light intensity was found to be therate-limiting factor for biosynthesis of (R)-3HB when the intensity wasbelow 60 μE/m²/s. When the light intensity was above 60 μE/m²/s, theacetoacetyl-CoA reductase activity was found to be the rate-limitingfactor. Expression of an additional copy of gene (phaB2_(eu-opt)) codingfor acetoacetyl-CoA reductase or enhancing the translation of gene phaB1by optimizing the RBS are both proven as effective strategies to enhancethe acetoacetyl-CoA reductase enzyme activity (Table 7), resulting in1.5- and 2.2-fold higher enzyme activity, respectively (Table 7). One ofthe engineered Synechocystis strains, R168, was able to produce andsecrete (R)-3HB to the extracellular culture medium at an average rateof ˜86 mg/L/day, with a peak productivity of 208 mg/L/day. Theeventually achieved cumulative titer of ˜1.6 g/L (FIG. 20) is to datethe highest titer reported in photoautotrophic production of thehydroxyalkanoates, precursors for biodegradable plastics and finechemicals.

It is noteworthy that without adding any organic carbon sources into theculture medium, the titer of the secreted (R)-3HB achieved in thisstudy, ˜30% dry cell weight equivalent, has reached the same level aswhat reported previously on cyanobacterial mixotrophic production ofPHB, intracellular granules that are non-secretable (Takahashi et al.,1998; Panda and Mallick, 2007). The technology developed here thus notonly has decreased the expense on culture feedstock, but also hasavoided the energy-expensive cell lysis process that is necessary priorto PHB recovery, leading to an economically more desirable and “greener”technology in microbial production of (R)-3HB. Additionally, it has beendemonstrated in this study that expression of genes of interest could befine-tuned from aspects including the gene copy number, transcription,translation, light intensity and CO₂ supply, which are critical toimproving the photosynthetic production of (R)-3HB in cyanobacteriumSynechocystis. These strategies are applicable to improving thephotosynthetic production of other chemicals in cyanobacteria.

All embodiments of any aspect of the invention can be combined withother embodiments of any aspect of the invention unless the contextclearly dictates otherwise.

Various changes in the details and components that have been describedmay be made by those skilled in the art within the principles and scopeof the invention herein described in the specification and defined inthe appended claims. Therefore, while the embodiments that have beenshown and described herein in what is believed to be the most practicaland preferred embodiments, it is recognized that departures can be madetherefrom within the scope of the invention, which is not to be limitedto the details disclosed herein but is to be accorded the full scope ofthe claims so as to embrace any and all equivalent processes andproducts.

REFERENCES

-   Agrawal, G. K., Kato, H., Asayama, M., Shirai, M., 2001. An AU-box    motif upstream of the SD sequence of light-dependent psbA    transcripts confers mRNA instability in darkness in cyanobacteria.    Nucleic Acids Res. 29, 1835-1843.-   Angermayr, S. A., Hellingwerf, K. J., 2013. On the use of metabolic    control analysis in the optimization of cyanobacterial biosolar cell    factories. J. Phys. Chem. B 117, 11169-11175.-   Angermayr, S. A., Paszota, M., Hellingwerf, K. J., 2012. Engineering    a cyanobacterial cell factory for production of lactic acid. Appl.    Environ. Microbiol. 78, 7098-7106.-   Dexter, J., Fu, P., 2009. Metabolic engineering of cyanobacteria for    ethanol production. Energ. Environ. Sci. 2, 857-864.-   Elhai, J., 1993. Strong and regulated promoters in the    cyanobacterium Anabaena PCC 7120. FEMS Microbiol. Lett. 114,    179-184.-   Eriksson, J., Salih, G. F., Ghebramedhin, H., Jansson, C., 2000.    Deletion mutagenesis of the 5′ psbA2 region in Synechocystis 6803:    identification of a putative cis element involved in    photoregulation. Mol. Cell Biol. Res. Commun. 3, 292-198.-   Gao, W., Zhang, W., Meldrum, D. R., 2011. RT-qPCR based quantitative    analysis of gene expression in single bacterial cells. J. Microbiol.    Methods 85, 221-227.-   Gao, Z., Zhao, H., Li, Z., Tan, X. and Lu, X., 2012. Photosynthetic    production of ethanol from carbon dioxide in genetically engineered    cyanobacteria. Energy Environ. Sci. 5, 9857-9865.-   Golden, S. S., 1995. Light-responsive gene expression in    cyanobacteria. J. Bacteriol. 177, 1651-1654.-   Guerrero, F., Carbonell, V., Cossu, M., Correddu, D., Jones, P.    R., 2012. Ethylene synthesis and regulated expression of recombinant    protein in Synechocystis sp. PCC 6803. PLoS ONE 7, e50470.-   Horie, Y., Ito, Y., Ono, M., Moriwaki, N., Kato, H., Hamakubo, Y.,    Amano, T., Wachi, M., Shirai, M., Asayama, M., 2007. Dark-induced    mRNA instability involves RNase E/G-type endoribonuclease cleavage    at the AU-box and SD sequences in cyanobacteria. Mol. Genet.    Genomics 278, 331-346.-   Huang, H. H., Camsund, D., Lindblad, P., Heidorn, T., 2010. Design    and characterization of molecular tools for a Synthetic Biology    approach towards developing cyanobacterial biotechnology. Nucleic    Acids Res. 38, 2577-2593.-   Liu, X., Sheng, J., Curtiss, R. 3rd., 2011. Fatty acid production in    genetically modified cyanobacteria. Proc. Natl. Acad. Sci. U.S.A.    108, 6899-6904.-   Ludwig, M., Bryant, D. A., 2011. Transcription profiling of the    model cyanobacterium Synechococcus sp. strain PCC 7002 by Next-Gen    (SOLiD™) Sequencing of cDNA. Front. Microbiol. 2, 41.-   Makrides, S. C., 1996. Strategies for achieving high-level    expression of genes in Escherichia coli. Microbiol. Rev. 60,    512-538.-   Marraccini, P., Bulteau, S., Cassier-Chauvat, C., Mermet-Bouvier,    P., Chauvat, F., 1993. A conjugative plasmid vector for promoter    analysis in several cyanobacteria of the genera Synechococcus and    Synechocystis. Plant Mol. Biol. 23, 905-909.-   Masamune, S., Walsh, C. T., Sinskey A. J. and Peoples, O. P., 1989.    Poly-(R)-3-hydroxybutyrate (PHB) biosynthesis: mechanistic studies    on the biological Claisen condensation catalyzed by β-ketoacyl    thiolase. Pure Appl. Chem., 61, 303-312.-   Mohamed, A., Jansson, C., 1989. Influence of light on accumulation    of photosynthesis-specific transcripts in the cyanobacterium    Synechocystis 6803. Plant Mol. Biol. 13, 693-700.-   Mohamed, A., Eriksson, J., Osiewacz, H. D., Jansson, C., 1993.    Differential expression of the psbA genes in the cyanobacterium    Synechocystis 6803. Mol. Gen. Genet. 238, 161-168.-   Nair, U., Thomas, C., Golden, S. S., 2001. Functional elements of    the strong psbAI promoter of Synechococcus elongatus PCC 7942. J.    Bacteriol. 183, 1740-1747.-   Niederholtmeyer, H., Wolfstadter, B., Savage, D., Silver, P., Way,    J., 2010. Engineering cyanobacteria to synthesize and export    hydrophilic products. Appl. Environ. Microb. 76, 3462-3466.-   Oliver, J. W., Machado, I. M., Yoneda, H., Atsumi, S., 2014.    Combinatorial optimization of cyanobacterial 2,3-butanediol    production. Metab. Eng. 22, 76-82.-   Panda, B., Mallick, N., 2007. Enhanced poly-beta-hydroxybutyrate    accumulation in a unicellular cyanobacterium, Synechocystis sp.    PCC 6803. Lett. Appl. Microbiol. 44, 194-198.-   Qi, F., Yao, L., Tan, X., Lu, X., 2013. Construction,    characterization and application of molecular tools for metabolic    engineering of Synechocystis sp. Biotechnol. Lett. 35, 1655-1661.-   Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M.,    Stanier, R. Y., 1979. Generic assignments, strain histories and    properties of pure cultures of cyanobacteria. J. Gen. Microbiol.    111, 1-61.-   Sakurai, I., Stazic, D., Eisenhut, M., Vuorio, E., Steglich, C.,    Hess, W. R., Aro, E. M., 2012. Positive regulation of psbA gene    expression by cis-encoded antisense RNAs in Synechocystis sp.    PCC 6803. Plant Physiol. 160, 1000-1010.-   Salis, H. M., Mirsky, E. A., Voigt, C. A., 2009. Automated design of    synthetic ribosome binding sites to control protein expression. Nat.    Biotechnol. 27, 946-950.-   Shen, C. R., Lan, E. I., Dekishima, Y., Baez, A., Cho, K. M.,    Liao, J. C., 2011. Driving forces enable high-titer anaerobic    1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol.    77, 2905-2915.-   Summerfield, T., Sherman, L., 2008. Global transcriptional response    of the alkali-tolerant cyanobacterium Synechocystis sp. strain PCC    6803 to a pH 10 environment. Appl. Environ. Microbiol. 74,    5276-5284.-   Takahashi, H., Miyake, M., Tokiwa, Y., Asada, Y., 1998. Improved    accumulation of poly-3-hydroxybutyrate by a recombinant    cyanobacterium. Biotechnol. Lett. 20, 183-186.-   Tseng, H. C., Martin, C. H., Nielsen, D. R., Prather, K. L., 2009.    Metabolic engineering of Escherichia coli for enhanced production of    (R)- and (S)-3-hydroxybutyrate. Appl. Environ. Microbiol. 75,    3137-3145.-   Ungerer, J., Tao, L., Davis, M., Ghirardi, M., Maness, P. C. and Yu,    J., 2012. Sustained photosynthetic conversion of CO₂ to ethylene in    recombinant cyanobacterium Synechocystis 6803. Energy Environ. Sci.    5, 8998-9006.-   Wang, B., Wang, J., Zhang, W., Meldrum, D. R., 2012. Application of    synthetic biology in cyanobacteria and algae. Front. Microbiol. 3,    344.-   Wang, B., Pugh, S., Nielsen, D. R., Zhang, W., Meldrum, D. R., 2013.    Engineering cyanobacteria for photosynthetic production of    3-hydroxybutyrate directly from CO₂. Metab. Eng. 16, 68-77.-   Wang, B., Pugh, S., Nielsen, D. R., Zhang, W., Meldrum, D. R., 2014.    Corrigendum to “Engineering cyanobacteria for photosynthetic    production of 3-hydroxybutyrate directly from CO₂” [Metab. Eng.    16 (2013) 68-77]. Metab. Eng. 21, 1.-   Williams, P. J. le B. and Laurensa, L. M. L. Microalgae as biodiesel    & biomass feedstocks: Review & analysis of the biochemistry,    energetics & economics. Energy Environ. Sci. 3, 554-590.-   Xu, Y., Alvey, R. M., Byrne, P. O., Graham, J. E., Shen, G.,    Bryant, D. A. Expression of genes in cyanobacteria: Adaptation of    endogenous plasmids as platforms for high-level gene expression in    Synechococcus sp. PCC 7002. In: Carpentier, R., ed. Photosynthesis    Research Protocols. New York, N.Y.: Springer, 2011:684.-   Yang, T., Lyons, S., Aguilar, C., Cuhel, R., Teske, A., 2011.    Microbial communities and chemosynthesis in yellowstone lake    sublacustrine hydrothermal vent waters. Front. Microbiol. 2, 130.

We claim:
 1. A method of photosynthetic production of 3-hydroxybutyratecomprising culturing a cyanobacterium Synechocystis strain geneticallyengineered to affect 3-hydroxybutyrate synthetic pathways in saidstrain.
 2. The method of claim 1, wherein solar energy is the soleenergy source during said culturing.
 8. The method of claim 1, whereinbicarbonate or CO2 is the sole carbon source during said culturing. 3.The method of claim 1, wherein carbonate is the sole carbon sourceduring said culturing.
 4. The method of claim 1, additionally comprisingthe step of multi-cycle production of 3-hydroxybutyrate by culturemedium exchange.
 5. The method of claim 1, wherein 3-hydroxybutyrateproduction titers exceed about 100 mg/l of Synechocystis culture.
 6. Themethod of claim 1, wherein 3-hydroxybutyrate production titers exceedabout 200 mg/l of Synechocystis culture.
 7. The method of claim 1,wherein the 3-hydroxybutyrate is secreted.
 8. The method of claim 1,wherein said cyanobacterium is Synechocystis sp. PCC
 6803. 9. The methodof claim 1, wherein one or more of the genes phaA2, phaB2, phaA, phaB,thil, hbd, tesB, phaC and phaE have been genetically engineered.